33 results on '"Kohki Yoshimoto"'
Search Results
2. Autophagy triggered by iron‐mediated <scp>ER</scp> stress is an important stress response to the early phase of Pi starvation in plants
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Yushi Yoshitake, Daiki Shinozaki, and Kohki Yoshimoto
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Membrane Lipids ,Iron ,Autophagy ,Genetics ,Cell Biology ,Plant Science ,Endoplasmic Reticulum ,Endoplasmic Reticulum Stress - Abstract
Inorganic phosphate (Pi) is essential for plant growth. However, Pi is often limiting in soil. Hence, plants have established several mechanisms of response to Pi starvation. One of the important mechanisms is Pi recycling, which includes membrane lipid remodeling and plastid DNA degradation via catabolic enzymes. However, the involvement of other degradation systems in Pi recycling remains unclear. Autophagy, a system for degradation of intracellular components, contributes to recycling of some nutrients, such as nitrogen, carbon, and zinc, under starvation. In the present study, we found that autophagy-deficient mutants depleted Pi early and exhibited severe leaf growth defects under Pi starvation. The main cargo of autophagy induced by early Pi depleted conditions was the endoplasmic reticulum (ER), indicating that ER-phagy, a type of autophagy that selectively degrades the ER, is involved in the response to the early phase of Pi starvation for contribution to Pi recycling. This ER-phagy was suppressed in an INOSITOL-REQUIRING ENZYME 1 double mutant, ire1a ire1b, in which ER stress responses are defective, suggesting that the early Pi starvation induced ER-phagy is induced by ER stress. Furthermore, iron limitation and inhibition of lipid-reactive oxygen species accumulation suppressed the ER-phagy. Interestingly, membrane lipid remodeling, a response to late Pi starvation, was accelerated in the ire1a ire1b under early Pi-depleted conditions. Our findings reveal the existence of two different phases of responses to Pi starvation (i.e. early and late) and indicate that ER stress-mediated ER-phagy is involved in Pi recycling in the early phase to suppress acceleration of the late phase.
- Published
- 2022
3. Seed dormancy 4 like1 of Arabidopsis is a key regulator of phase transition from embryo to vegetative development
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Lipeng Zheng, Masahiko Otani, Yuri Kanno, Mitsunori Seo, Yushi Yoshitake, Kohki Yoshimoto, Kazuhiko Sugimoto, and Naoto Kawakami
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Arabidopsis Proteins ,Gene Expression Regulation, Plant ,Seedlings ,Seeds ,Genetics ,Arabidopsis ,Germination ,Oryza ,Cell Biology ,Plant Science ,Plant Dormancy - Abstract
Seed dormancy is an adaptive trait that enables plants to survive adverse conditions and restart growth in a season and location suitable for vegetative and reproductive growth. Control of seed dormancy is also important for crop production and food quality because it can help induce uniform germination and prevent preharvest sprouting. Rice preharvest sprouting quantitative trait locus analysis has identified Seed dormancy 4 (Sdr4) as a positive regulator of dormancy development. Here, we analyzed the loss-of-function mutant of the Arabidopsis ortholog, Sdr4 Like1 (SFL1), and found that the sfl1-1 seeds showed precocious germination at the mid- to late-maturation stage similar to rice sdr4 mutant, but converted to become more dormant than the wild type during maturation drying. Coordinated with the dormancy levels, expression levels of the seed maturation and dormancy master regulator genes, ABI3, FUS3, and DOG1 in sfl1-1 seeds were lower than in wild type at early- and mid-maturation stages, but higher at the late-maturation stage. In addition to the seed dormancy phenotype, sfl1-1 seedlings showed a growth arrest phenotype and heterochronic expression of LAFL (LEC1, ABI3, FUS3, LEC2) and DOG1 in the seedlings. These data suggest that SFL1 is a positive regulator of initiation and termination of the seed dormancy program. We also found genetic interaction between SFL1 and the SFL2, SFL3, and SFL4 paralogs of SFL1, which impacts on the timing of the phase transition from embryo maturation to seedling growth.
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- 2022
4. Autophagy balances the zinc–iron seesaw caused by Zn-stress
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Daiki Shinozaki and Kohki Yoshimoto
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inorganic chemicals ,biology ,Iron ,Autophagy ,Arabidopsis ,chemistry.chemical_element ,Plant Science ,Zinc ,biology.organism_classification ,Cell biology ,chemistry ,Seesaw molecular geometry ,Gene Expression Regulation, Plant ,Arabidopsis thaliana ,Homeostasis ,Intracellular - Abstract
Under zinc (Zn) deficiency, plants take up excess iron (Fe), but the uptake is inhibited under Zn excess. Coordination between intracellular recycling, transport, and sensing is essential for Zn–Fe homeostasis. A new study shows that autophagy resupplies Zn2+ and Fe2+ to correct intracellular Zn–Fe imbalances.
- Published
- 2021
5. Optimal Distribution of Iron to Sink Organs via Autophagy Is Important for Tolerance to Excess Zinc in Arabidopsis
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Kohki Yoshimoto, Daiki Shinozaki, and Keitaro Tanoi
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Chlorophyll ,Physiology ,Iron ,ATG5 ,Arabidopsis ,chemistry.chemical_element ,Plant Science ,Zinc ,Gene Expression Regulation, Plant ,Stress, Physiological ,Autophagy ,Arabidopsis thaliana ,Chlorosis ,biology ,Chemistry ,food and beverages ,Cell Biology ,General Medicine ,Iron Deficiencies ,Meristem ,biology.organism_classification ,Cell biology ,Plant Leaves ,Sink (computing) ,Reactive Oxygen Species - Abstract
Zinc (Zn) is nutritionally an essential metal element, but excess Zn in the environment is toxic to plants. Autophagy is a major pathway responsible for intracellular degradation. Here, we demonstrate the important role of autophagy in adaptation to excess Zn stress. We found that autophagy-defective Arabidopsis thaliana (atg2 and atg5) exhibited marked excess Zn-induced chlorosis and growth defects relative to wild-type (WT). Imaging and biochemical analyses revealed that autophagic activity was elevated under excess Zn. Interestingly, the excess Zn symptoms of atg5 were alleviated by supplementation of high levels of iron (Fe) to the media. Under excess Zn, in atg5, Fe starvation was especially severe in juvenile true leaves. Consistent with this, accumulation levels of Fe3+ near the shoot apical meristem remarkably reduced in atg5. Furthermore, excision of cotyledons induced severe excess Zn symptoms in WT, similar to those observed in atg5. Our data suggest that Fe3+ supplied from source leaves (cotyledons) via autophagy is distributed to sink leaves (true leaves) to promote healthy growth under excess Zn, revealing a new dimension, the importance of heavy-metal stress responses by the intracellular recycling.
- Published
- 2020
6. Pexophagy Protects Plants from Reactive Oxygen Species-induced Damage under High-intensity Light
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Akira Kato, Kazusato Oikawa, Shino Goto-Yamada, Keisuke Shimoda, Kenji Yamada, Atsushi Takemiya, Matsuo Uemura, Kohki Yoshimoto, Ikuko Hara-Nishimura, Keiji Numata, Yasuko Hayashi, Shoji Mano, Mikio Nishimura, Daisuke Takahashi, Yoshinori Ohsumi, Maki Kondo, Michitaro Shibata, Yoshitaka Kimori, and Haruko Ueda
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chemistry.chemical_classification ,High intensity light ,Reactive oxygen species ,Chemistry ,Biophysics - Abstract
Light is essential for photosynthesis, but it has the potential to elevate intracellular levels of reactive oxygen species (ROS) during photosynthesis. Photorespiration is a metabolic pathway in photosynthesis to metabolise oxidised by products from chloroplasts, and it generates a high level of ROS in peroxisomes. Since high levels of ROS are toxic, plants must manage damage from ROS. However, the cellular mechanism to elude leaf damage from ROS in peroxisomes is not fully explored. Here we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We found that a series of peup mutants, which is a defect in autophagy degradation of peroxisomes, showed light-intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates were specifically engulfed by pre-autophagosomal structures and vacuolar membranes, but they were not degraded in the mutants. ATG18a-GFP and GFP-2 × FYVE, which both bind to phosphatidylinositol 3-phosphate, preferentially targeted the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulated cells under high-intensity light conditions. Our findings provide new information to better understand the plant stress response caused by light irradiation.
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- 2020
7. Autophagy and Nutrients Management in Plants
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Fabien Chardon, Mathieu Pottier, Michèle Reisdorf-Cren, Daiki Shinozaki, Sébastien Thomine, Kohki Yoshimoto, Céline Masclaux-Daubresse, Marien Havé, Jie Luo, Anne Marmagne, and Qinwu Chen
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0106 biological sciences ,0301 basic medicine ,Nutrient cycle ,leaf senescence ,Plant Development ,Review ,Biology ,01 natural sciences ,nitrogen use efficiency ,03 medical and health sciences ,iron ,Nutrient ,Stress, Physiological ,Autophagy ,Plant Proteins ,2. Zero hunger ,plant proteases ,business.industry ,zinc ,fungi ,food and beverages ,nitrogen remobilization ,Nutrients ,General Medicine ,Plant engineering ,Plants ,15. Life on land ,Biotechnology ,030104 developmental biology ,Metabolic Engineering ,Seeds ,business ,010606 plant biology & botany - Abstract
Nutrient recycling and mobilization from organ to organ all along the plant lifespan is essential for plant survival under changing environments. Nutrient remobilization to the seeds is also essential for good seed production. In this review, we summarize the recent advances made to understand how plants manage nutrient remobilization from senescing organs to sink tissues and what is the contribution of autophagy in this process. Plant engineering manipulating autophagy for better yield and plant tolerance to stresses will be presented.
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- 2019
8. Unveiling the molecular mechanisms of plant autophagy – from autophagosomes to vacuoles in plants
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Kohki Yoshimoto and Yoshinori Ohsumi
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0301 basic medicine ,biology ,Physiology ,Saccharomyces cerevisiae ,Autophagy ,Autophagosomes ,food and beverages ,Cell Biology ,Plant Science ,General Medicine ,Vacuole ,biology.organism_classification ,Phenotype ,Yeast ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Plant Cells ,Vacuoles ,Identification (biology) ,Gene ,Intracellular ,Plant Proteins - Abstract
Autophagy is an evolutionarily conserved intracellular vacuolar process. Since Christian de Duve first coined the term 'autophagy' in 1963, it had not been well understood at the molecular level until much later, due to limitations in biochemical approaches and/or morphological approaches posed by electron microscopy. An important milestone was achieved with the isolation and identification of autophagy-related (ATG) genes by genetic screening using yeast Saccharomyces cerevisiae. ATG genes are well conserved in most eukaryotic organisms, which allowed the subsequent isolation of ATG gene-knockouts in plants. From the phenotypic analyses of the autophagy-defective plants, the physiological roles of autophagy have been predicted. However, in some cases, all the phenotypes cannot be simply explained by defects in autophagy. Therefore, in order to fully understand the physiological implications of plant autophagy, it is quite important to elucidate the molecular mechanisms involved in each process in macro-/micro-autophagy. Although, until recently, our understanding of the molecular mechanisms of plant autophagy was lagging compared to similar research in yeast and animals, current studies have made many great advances in the plant research field. In this review, we discuss current knowledge of the molecular mechanisms of plant autophagy, from autophagy-induction/autophagosome-formation to vacuolar degradation, comparing these to processes in yeast and mammals. We also review aspects of plant autophagy research that require further investigation in the future.
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- 2018
9. Autophagy, plant senescence, and nutrient recycling
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Liliana Avila-Ospina, Céline Masclaux-Daubresse, Kohki Yoshimoto, Michaël Moison, Institut Jean-Pierre Bourgin (IJPB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, CETIOM (Centre Technique des Oleagineux), Biologie et Amelioration des Plantes, INRA, INRA Package programme, European Project: 264394, Avila-Ospina, Liliana, and Moison, Michaël
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0106 biological sciences ,Senescence ,proteolysis ,Chloroplasts ,Nitrogen ,Physiology ,[SDV]Life Sciences [q-bio] ,Proteolysis ,media_common.quotation_subject ,Cell ,Flowers ,Plant Science ,Biology ,proteolyse ,01 natural sciences ,03 medical and health sciences ,Autophagy ,medicine ,vesicular trafficking ,Organism ,030304 developmental biology ,media_common ,Plant senescence ,Nitrogen remobilization ,Rubisco containing bodies ,plant cell death ,0303 health sciences ,medicine.diagnostic_test ,Longevity ,Plants ,Cell biology ,Plant Leaves ,medicine.anatomical_structure ,Adaptation ,010606 plant biology & botany - Abstract
Large numbers of publications have appeared over the last few years, dealing with the molecular details of the regulation and process of the autophagy machinery in animals, plants, and unicellular eukaryotic organisms. This strong interest is caused by the fact that the autophagic process is involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. In plants, as in other eukaryotes, autophagy is associated with longevity as mutants display early and strong leaf senescence symptoms, however, the exact role of autophagy as a pro-survival or pro-death process is unclear. Recently, evidence that autophagy participates in nitrogen remobilization has been provided, but the duality of the role of autophagy in leaf longevity and/or nutrient recycling through cell component catabolism remains. This review aims to give an overview of leaf senescence-associated processes from the physiological point of view and to discuss relationships between nutrient recycling, proteolysis, and autophagy. The dual role of autophagy as a pro-survival or pro-death process is discussed.
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- 2014
10. [Untitled]
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Kohki YOSHIMOTO
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- 2014
11. Autophagy machinery controls nitrogen remobilization at the whole‐plant level under both limiting and ample nitrate conditions in Arabidopsis
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Céline Masclaux-Daubresse, Fabienne Soulay, Anne Guiboileau, Marie-Paule Bataillé, Jean-Christophe Avice, Kohki Yoshimoto, Institut Jean-Pierre Bourgin (IJPB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Ecophysiologie Végétale, Agronomie et Nutritions (EVA), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Recherche Agronomique (INRA), SAKURA [21124QA], French Ministere des Affaires Etrangeres et Europeennes, Japan Society for the Promotion of Science, Institut Jean-Pierre Bourgin ( IJPB ), Institut National de la Recherche Agronomique ( INRA ) -AgroParisTech, Ecophysiologie Végétale, Agronomie et Nutritions ( EVA ), Université de Caen Normandie ( UNICAEN ), and Normandie Université ( NU ) -Normandie Université ( NU ) -Institut National de la Recherche Agronomique ( INRA )
- Subjects
[SDV.SA]Life Sciences [q-bio]/Agricultural sciences ,0106 biological sciences ,Physiology ,Mutant ,Arabidopsis ,NATURAL VARIATION ,Plant Science ,Vacuole ,01 natural sciences ,Autophagy-Related Protein 5 ,chemistry.chemical_compound ,Nitrate ,CHLOROPLASTS ,Biomass ,[ SDV.SA ] Life Sciences [q-bio]/Agricultural sciences ,harvest index (HI) ,Cellular Senescence ,2. Zero hunger ,0303 health sciences ,biology ,15N ,food and beverages ,nitrogen remobilization ,Plants, Genetically Modified ,Chloroplast ,Phenotype ,Seeds ,RNA Interference ,RIBULOSE-1 ,autophagy ,Nitrogen ,PROTEINS ,Ribulose-Bisphosphate Carboxylase ,03 medical and health sciences ,Botany ,LEAVES ,LEAF SENESCENCE ,Nitrogen cycle ,030304 developmental biology ,Nitrates ,Nitrogen Isotopes ,THALIANA ,Arabidopsis Proteins ,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE ,Autophagy ,RuBisCO ,DEGRADATION ,yield ,biology.organism_classification ,RUBISCO ,Carbon ,Phosphoric Monoester Hydrolases ,Plant Leaves ,chemistry ,Mutation ,Vacuoles ,biology.protein ,nitrogen use efficiency (NUE) ,STARVATION ,010606 plant biology & botany - Abstract
• Processes allowing the recycling of organic nitrogen and export to young leaves and seeds are important determinants of plant yield, especially when plants are nitrate-limited. Because autophagy is induced during leaf ageing and in response to nitrogen starvation, its role in nitrogen remobilization was suspected. It was recently shown that autophagy participates in the trafficking of Rubisco-containing bodies to the vacuole. • To investigate the role of autophagy in nitrogen remobilization, several autophagy-defective (atg) Arabidopsis mutants were grown under low and high nitrate supplies and labeled with at the vegetative stage in order to determine (15) N partitioning in seeds at harvest. Because atg mutants displayed earlier and more rapid leaf senescence than wild type, we investigated whether their defects in nitrogen remobilization were related to premature leaf cell death by studying the stay-green atg5.sid2 and atg5.NahG mutants. • Results showed that nitrogen remobilization efficiency was significantly lower in all the atg mutants irrespective of biomass defects, harvest index reduction, leaf senescence phenotypes and nitrogen conditions. • We conclude that autophagy core machinery is needed for nitrogen remobilization and seed filling.
- Published
- 2012
12. Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response inArabidopsis
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Yoshinori Ohsumi, Ken Shirasu, Kohki Yoshimoto, Yuji Kamiya, Chiara Consonni, Yusuke Jikumaru, Miyako Kusano, and Ralph Panstruga
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Senescence ,Programmed cell death ,Arabidopsis ,Cyclopentanes ,Plant Science ,Autophagy-Related Protein 5 ,chemistry.chemical_compound ,Plant Growth Regulators ,Gene Expression Regulation, Plant ,Autophagy ,Oxylipins ,Research Articles ,Innate immune system ,biology ,Arabidopsis Proteins ,Jasmonic acid ,Hydrogen Peroxide ,Cell Biology ,Ethylenes ,biology.organism_classification ,Phosphoric Monoester Hydrolases ,Cell biology ,chemistry ,RNA, Plant ,Signal transduction ,Salicylic Acid ,Intracellular ,Signal Transduction - Abstract
Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.
- Published
- 2009
13. OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice
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Jun-Hye Shin, Jong-Seong Jeon, Gynheung An, Yoshinori Ohsumi, and Kohki Yoshimoto
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DNA, Bacterial ,Paraquat ,Autophagosome ,DNA, Complementary ,Cell Survival ,Molecular Sequence Data ,Mutant ,GUS reporter system ,Sodium Chloride ,Biology ,Genes, Plant ,Gene Expression Regulation, Plant ,Phagosomes ,Gene expression ,Organelle ,Autophagy ,Amino Acid Sequence ,Molecular Biology ,Gene ,Vascular tissue ,Glucuronidase ,Plant Proteins ,Gene Expression Profiling ,Oryza ,Cell Biology ,General Medicine ,Mutagenesis, Insertional ,Oxidative Stress ,Protein Transport ,Biochemistry ,Mutation ,Subcellular Fractions - Abstract
Autophagy degrades toxic materials and old organelles, and recycles nutrients in eukaryotic cells. Whereas the studies on autophagy have been reported in other eukaryotic cells, its functioning in plants has not been well elucidated. We analyzed the roles of OsATG10 genes, which are autophagy-related. Two rice ATG10 genes - OsATG10a and OsATG10b - share significant sequence homology (about 75%), and were ubiquitously expressed in all organs examined here. GUS assay indicated that OsATG10b was highly expressed in the mesophyll cells and vascular tissue of younger leaves, but its level of expression decreased in older leaves. We identified T-DNA insertional mutants in that gene. Those osatg10b mutants were sensitive to treatments with high salt and methyl viologen (MV). Monodansylcadaverine-staining experiments showed that the number of autophagosomes was significantly decreased in the mutants compared with the WT. Furthermore, the amount of oxidized proteins increased in MV-treated mutant seedlings. These results demonstrate that OsATG10b plays an important role in the survival of rice cells against oxidative stresses.
- Published
- 2009
14. Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by anATGGene-Dependent Autophagic Process
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Hiroyuki Ishida, Amane Makino, Yoshinori Ohsumi, Kohki Yoshimoto, Yuichi Yano, Maureen R. Hanson, Tadahiko Mae, Daniel Reisen, and Masanori Izumi
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biology ,Physiology ,Immunoelectron microscopy ,Vacuolar lumen ,fungi ,RuBisCO ,food and beverages ,Plant Science ,Vacuole ,Green fluorescent protein ,Chloroplast ,Biochemistry ,Plant protein ,Genetics ,biology.protein ,Chloroplast Proteins - Abstract
During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H+-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.
- Published
- 2008
15. The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy
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Kohki Yoshimoto, Yoshinori Ohsumi, Yuko Fujioka, Fuyuhiko Inagaki, and Nobuo Suzuki
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Models, Molecular ,Protein Folding ,Arabidopsis Proteins ,Molecular Sequence Data ,ATG5 ,Autophagy ,Arabidopsis ,Cell Biology ,Crystal structure ,Biology ,Conjugated system ,Crystallography, X-Ray ,Cell biology ,ATG12 ,Biochemistry ,Plant protein ,Amino Acid Sequence ,Hydrophobic and Hydrophilic Interactions ,Molecular Biology ,Conjugate - Abstract
Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 A resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.
- Published
- 2005
16. A Novel Selection Method Based on the Expression Level of Green Fluorescent Protein Measured with a Quantitative Fluorescence Imager
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Yasuo Niwa, Shingo Goto, Ryoko Kuruto-Niwa, and Kohki Yoshimoto
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Reporter gene ,Fluorescence-lifetime imaging microscopy ,Plant Science ,Biology ,Molecular biology ,Green fluorescent protein ,Serine ,Transformation (genetics) ,Quantitative fluorescence ,Biophysics ,Threonine ,Agronomy and Crop Science ,Function (biology) ,Biotechnology - Abstract
Green fluorescent protein (GFP) has been used extensively as a novel non-invasive reporter in the investigation of issues such as promoter function, transformation marker recognition, and sub-cellular localization analysis. A plant-optimized synthetic GFP modified via the replacement of the serine to threonine at position 65 [sGFP(S65T)] has achieved high levels of expression with no toxic side-effects in many plants. It would be useful to develop a non-invasive selection system based only upon GFP expression levels. A trapping vector was constructed using an sGFP(S65T) and a quantitative fluorescence imaging system was used for non-invasive screening of trapped tobacco cultured cells. Putative trapped lines could be identified using this system. Moreover, the GFP products from the candidate trapped lines could be quantitatively visualized directly via SDS-PAGE using the fluorescence imaging system. We conclude that the non-invasive selection system described here could be a powerful new tool for plant biotechnology.
- Published
- 2003
17. Quality control of plant peroxisomes in organ specific manner via autophagy
- Author
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Michitaro Shibata, Kiminori Toyooka, Mayuko Sato, Yoshinori Ohsumi, Mikio Nishimura, Kohki Yoshimoto, Kazusato Oikawa, Ken Shirasu, and Maki Kondo
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biology ,Autophagy ,Cell Biology ,Vacuole ,Peroxisome ,biology.organism_classification ,Cell biology ,Biochemistry ,Catalase ,Arabidopsis ,Organelle ,Glyoxysome ,biology.protein ,Arabidopsis thaliana - Abstract
Peroxisomes are essential organelles characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG) gene-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5 knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles by autophagy for degradation. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves for proper plant development under natural growth conditions.
- Published
- 2014
18. Comparison of Strength of Endogenous and Exogenous Gene Promoters in Arabidopsis Chloroplasts
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Kohki Yoshimoto, Kyoichi Isono, Mao Sakaiya, and Hirokazu Kobayashi
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Nuclear gene ,biology ,Operon ,food and beverages ,Promoter ,Plant Science ,Chimeric gene ,biology.organism_classification ,Molecular biology ,Chloroplast ,Chloroplast DNA ,Arabidopsis ,Agronomy and Crop Science ,Gene ,Biotechnology - Abstract
We have focused on possible stronger promoters in the chloroplast: those of psbA encoding D1 protein of photosystem II reaction center, 16S rDNA in rrn operon, the bacterial fused promoter tac, and the bacteriophage T7 gene φ10 in combination with transgenic T7 RNA polymerase (RNAP). Arabidopsis plants were made transgenic in the nuclear genome with the construct of a chimeric gene for T7 RNAP fused to a chloroplast transit peptide at its N-terminus placed under the control of CaMV 35S promoter. We have transiently expressed gene for β-glucuronidase (GUS) under control of the above promoters in the Arabidopsis chloroplast followed by particle bombardment. Expression in the chloroplast but not in the nucleus was confirmed histochemically and by treatment with α-amanitin. T7 promoter was the strongest among the examined promoters in the Arabidopsis chloroplast, being applicable to higher expression of foreign genes in the chloroplast with managed expression of T7 RNAP.
- Published
- 2001
19. Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ 70 factors of bacterial RNA polymerases in Arabidopsis thaliana
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Akiho Yokota, Kohki Yoshimoto, Hirokazu Kobayashi, Kyoichi Isono, Masanori Shimizu, Kimiyuki Satoh, and Yasuo Niwa
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Chloroplasts ,Molecular Sequence Data ,Arabidopsis ,Sigma Factor ,Genes, Plant ,Genome ,chemistry.chemical_compound ,Bacterial Proteins ,Transcription (biology) ,Sigma factor ,RNA polymerase ,Amino Acid Sequence ,Plastid ,Gene ,Polymerase ,Genetics ,Multidisciplinary ,biology ,Arabidopsis Proteins ,DNA-Directed RNA Polymerases ,Biological Sciences ,biology.organism_classification ,chemistry ,biology.protein ,Peptides ,Sequence Alignment - Abstract
Genes for σ-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1 , SIG2 , and SIG3 , for polypeptides possessing amino acid sequences for domains conserved in σ 70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana . Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.
- Published
- 1997
20. A possible involvement of autophagy in amyloplast degradation in columella cells during hydrotropic response of Arabidopsis roots
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Ken Shirasu, Hideyuki Takahashi, Yutaka Miyazawa, Mikio Nishimura, Nobuharu Fujii, Kenji Yamada, Hiroyuki Ishida, Mayumi Nakayama, Nahoko Higashitani, Kohki Yoshimoto, Yasuko Kaneko, and Shinya Wada
- Subjects
Autophagosome ,Time Factors ,Recombinant Fusion Proteins ,Gravitropism ,Arabidopsis ,Autophagy-Related Proteins ,Plant Science ,Hydrotropism ,Vacuole ,Endoplasmic Reticulum ,Plant Roots ,Tropism ,Microscopy, Electron, Transmission ,Gene Expression Regulation, Plant ,Stress, Physiological ,Botany ,Genetics ,Autophagy ,Amyloplast ,Plastids ,Cell Nucleus ,Microscopy, Confocal ,biology ,Dehydration ,Arabidopsis Proteins ,Endoplasmic reticulum ,Cell Polarity ,biology.organism_classification ,Plants, Genetically Modified ,Cell biology ,Vacuolization ,Seedlings ,Mutation ,Vacuoles ,Transcription Factors - Abstract
Seedling roots display not only gravitropism but also hydrotropism, and the two tropisms interfere with one another. In Arabidopsis (Arabidopsis thaliana) roots, amyloplasts in columella cells are rapidly degraded during the hydrotropic response. Degradation of amyloplasts involved in gravisensing enhances the hydrotropic response by reducing the gravitropic response. However, the mechanism by which amyloplasts are degraded in hydrotropically responding roots remains unknown. In this study, the mechanistic aspects of the degradation of amyloplasts in columella cells during hydrotropic response were investigated by analyzing organellar morphology, cell polarity and changes in gene expression. The results showed that hydrotropic stimulation or systemic water stress caused dramatic changes in organellar form and positioning in columella cells. Specifically, the columella cells of hydrotropically responding or water-stressed roots lost polarity in the distribution of the endoplasmic reticulum (ER), and showed accelerated vacuolization and nuclear movement. Analysis of ER-localized GFP showed that ER redistributed around the developed vacuoles. Cells often showed decomposing amyloplasts in autophagosome-like structures. Both hydrotropic stimulation and water stress upregulated the expression of AtATG18a, which is required for autophagosome formation. Furthermore, analysis with GFP-AtATG8a revealed that both hydrotropic stimulation and water stress induced the formation of autophagosomes in the columella cells. In addition, expression of plastid marker, pt-GFP, in the columella cells dramatically decreased in response to both hydrotropic stimulation and water stress, but its decrease was much less in the autophagy mutant atg5. These results suggest that hydrotropic stimulation confers water stress in the roots, which triggers an autophagic response responsible for the degradation of amyloplasts in columella cells of Arabidopsis roots.
- Published
- 2011
21. The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis
- Author
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Ohkmae K. Park, Soon Il Kwon, Hong Joo Cho, Jin Hee Jung, Ken Shirasu, and Kohki Yoshimoto
- Subjects
Programmed cell death ,biology ,Cell ,Autophagy ,Mutant ,Wild type ,food and beverages ,Cell Biology ,Plant Science ,biology.organism_classification ,Cell biology ,medicine.anatomical_structure ,Tracheary element differentiation ,Arabidopsis ,Genetics ,medicine ,Rab - Abstract
The tracheary elements (TEs) of the xylem serve as the water-conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1-301, and an autophagy mutant atg5-1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.
- Published
- 2010
22. The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis
- Author
-
Soon Il, Kwon, Hong Joo, Cho, Jin Hee, Jung, Kohki, Yoshimoto, Ken, Shirasu, and Ohkmae K, Park
- Subjects
Arabidopsis Proteins ,Gene Expression Regulation, Plant ,RNA, Plant ,Xylem ,rab GTP-Binding Proteins ,Gene Knockdown Techniques ,Arabidopsis ,Autophagy ,Cell Differentiation ,Plants, Genetically Modified - Abstract
The tracheary elements (TEs) of the xylem serve as the water-conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1-301, and an autophagy mutant atg5-1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.
- Published
- 2010
23. Physiological roles of autophagy in plants: does plant autophagy have a pro-death function?
- Author
-
Kohki Yoshimoto
- Subjects
Senescence ,Programmed cell death ,Cell Death ,Mutant ,Autophagy ,fungi ,food and beverages ,Plant Science ,Review ,Biology ,Plants ,Models, Biological ,Cell biology ,Phenotype ,Biochemistry ,Apoptosis ,Plant Cells ,Signal transduction ,Intracellular ,Function (biology) - Abstract
Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. Early morphological studies suggested that autophagy occurs in plant cells and predicted that autophagy has a variety of functions in plant growth and development. However, it is only since the identification of autophagy genes that the physiological roles of autophagy in plants have become apparent. Recent reverse genetic studies indicate that autophagy defects in higher plants result in early senescence and excessive immunity-related programmed cell death (PCD), irrespective of nutrient conditions, suggesting that plant autophagy has an important pro-survival function during these types of cell death. Further biochemical and pharmacological studies in combination with double mutant analyses revealed that excessive salicylic acid (SA) signaling is a major factor in autophagy-defective plant-dependent cell death and that the SA signal can induce autophagy. These results demonstrate a novel physiological function for plant autophagy that operates a negative feedback loop to modulate SA signaling.
- Published
- 2010
24. Chloroplasts autophagy during senescence of individually darkened leaves
- Author
-
Amane Makino, Masanori Izumi, Tadahiko Mae, Kohki Yoshimoto, Hiroyuki Ishida, Shinya Wada, and Yoshinori Ohsumi
- Subjects
Chlorophyll ,DNA, Bacterial ,Chloroplasts ,Physiology ,Ribulose-Bisphosphate Carboxylase ,Population ,Arabidopsis ,Plant Science ,Vacuole ,Photosynthesis ,chemistry.chemical_compound ,Botany ,Autophagy ,Genetics ,Arabidopsis thaliana ,education ,Cellular Senescence ,Plant Proteins ,education.field_of_study ,biology ,Vacuolar lumen ,RuBisCO ,fungi ,food and beverages ,biology.organism_classification ,Article Addendum ,Cell biology ,Plant Leaves ,Chloroplast ,Mutagenesis, Insertional ,Cytosol ,chemistry ,Biochemistry ,biology.protein ,Chloroplast Proteins ,Cell aging ,Research Article - Abstract
We recently reported that autophagy plays a role in chloroplasts degradation in individually-darkened senescing leaves. Chloroplasts contain approximately 80% of total leaf nitrogen, mainly as photosynthetic proteins, predominantly ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco). During leaf senescence, chloroplast proteins are degraded as a major source of nitrogen for new growth. Concomitantly, while decreasing in size, chloroplasts undergo transformation to non-photosynthetic gerontoplasts. Likewise, over time the population of chloroplasts (gerontoplasts) in mesophyll cells also decreases. While bulk degradation of the cytosol and organelles is mediated by autophagy, the role of chloroplast degradation is still unclear. In our latest study, we darkened individual leaves to observe chloroplast autophagy during accelerated senescence. At the end of the treatment period chloroplasts were much smaller in wild-type than in the autophagy defective mutant, atg4a4b-1, with the number of chloroplasts decreasing only in wild-type. Visualizing the chloroplast fractions accumulated in the vacuole, we concluded that chloroplasts were degraded by two different pathways, one was partial degradation by small vesicles containing only stromal-component (Rubisco containing bodies; RCBs) and the other was whole chloroplast degradation. Together, these pathways may explain the morphological attenuation of chloroplasts during leaf senescence and describe the fate of chloroplasts.
- Published
- 2009
25. Role of chloroplasts and other plastids in ageing and death of plants and animals: a tale of Vishnu and Shiva
- Author
-
Wouter G. van Doorn and Kohki Yoshimoto
- Subjects
Aging ,Chloroplasts ,Photosynthesis ,Biochemistry ,Algae ,Chromoplast ,Botany ,Animals ,Plastid ,Symbiosis ,Molecular Biology ,Cellular Senescence ,Plant Physiological Phenomena ,Apicoplast ,Endosymbiosis ,biology ,Cell Death ,fungi ,food and beverages ,Eukaryota ,biology.organism_classification ,Anthozoa ,Chloroplast ,Neurology ,Cell aging ,Biotechnology - Abstract
Chloroplasts (chlorophyll-containing plastids) and other plastids are found in all plants and many animals. They are crucial to the survival of plants and most of the animals that harbour them. An example of a non-photosynthesizing plastid in animals is the apicoplast in the malaria-causing Plasmodium species, which is required for survival of the parasite. Many animals (such as sea slugs, sponges, reef corals, and clams) consume prey containing chloroplasts, or feed on algae. Some of these incorporate the chloroplasts from their food, or whole algal cells, into their own cells. Other species from these groups place algal cells between their own cells. Reef-building corals often lose their intracellular algae as a result of environmental changes, resulting in coral bleaching and death. The sensitivity of the chloroplast internal membranes to temperature stress is one of the reasons for coral death. Chloroplasts can also be a causal factor in the processes leading to whole-plant death, as the knockout of a gene encoding a chloroplast protein delayed the yellowing that proceeds death in tobacco plants. It is concluded that chloroplasts and other plastids are essential to individual survival in many species, including animals, and that they also play a role in triggering death in some plant and animal species.
- Published
- 2009
26. Visualization of Rubisco-Containing Bodies Derived from Chloroplasts in Living Cells of Arabidopsis
- Author
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Tadahiko Mae, Amane Makino, Yoshinori Ohsumi, Daniel Reisen, Hiroyuki Ishida, Kohki Yoshimoto, and Maureen R. Hanson
- Subjects
biology ,Chemistry ,Immunoelectron microscopy ,fungi ,RuBisCO ,food and beverages ,Vacuole ,biology.organism_classification ,Green fluorescent protein ,Chloroplast ,Biochemistry ,Plant protein ,Arabidopsis ,Botany ,biology.protein ,Chlorophyll fluorescence - Abstract
During senescence and times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco). Previously by immunoelectron microscopy (IEM), we demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves (Chiba et al. 2003). In this study, we visualized RCBs in living cells of transgenic Arabidopsis plants containing stroma-targeted green fluorescent protein (GFP). When leaves of transgenic Arabidopsis plants were incubated under starvation conditions with a vacuolar-ATPase inhibitor, spherical bodies exhibiting GFP fluorescence without chlorophyll fluorescence were observed. Spherical bodies were not observed when leaves were provided with a sugar and nutrient solution. IEM of concanamycin-A-treated leaves with anti-Rubisco antibodies confirmed the existence of RCBs in the vacuolar compartment. These results suggest the hypothesis that stromal proteins can be mobilized to the vacuole via RCBs by possibly autophagy for the degradation.
- Published
- 2008
27. In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy
- Author
-
Kohki Yoshimoto, Yuko Fujioka, Nobuo N. Noda, Fuyuhiko Inagaki, Yoshinori Ohsumi, and Kiyonaga Fujii
- Subjects
Mammals ,biology ,Arabidopsis Proteins ,Ubiquitin ,ATG8 ,Phosphatidylethanolamines ,Saccharomyces cerevisiae ,Arabidopsis ,Ubiquitination ,Cell Biology ,biology.organism_classification ,Biochemistry ,Yeast ,In vitro ,ATG12 ,Autophagy ,Arabidopsis thaliana ,Animals ,Molecular Biology ,Autophagy-Related Protein 12 ,Conjugate - Abstract
Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b–8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.
- Published
- 2007
28. AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells
- Author
-
Masaki Hattori, Takao Suzuki, Yuji Moriyasu, Yuko Inoue, Yoshinori Ohsumi, and Kohki Yoshimoto
- Subjects
Sucrose ,Physiology ,Mutant ,Meristem ,Arabidopsis ,Autophagy-Related Proteins ,Plant Science ,Vacuole ,medicine.disease_cause ,Genes, Plant ,Aminopeptidases ,Autophagy-Related Protein 5 ,Gene Expression Regulation, Plant ,medicine ,Autophagy ,Gene ,Mutation ,biology ,Arabidopsis Proteins ,Adenine ,Cell Biology ,General Medicine ,biology.organism_classification ,Phosphoric Monoester Hydrolases ,Cell biology ,Biochemistry ,Cytoplasm ,Vacuoles - Abstract
In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.
- Published
- 2006
29. [Recent advances in plant autophagy research: plant atg mutants]
- Author
-
Kohki, Yoshimoto
- Subjects
Saccharomyces cerevisiae Proteins ,Plant Cells ,Mutation ,Vacuoles ,Autophagy ,Plant Physiological Phenomena ,Plant Proteins - Published
- 2006
30. Plant autophagy puts the brakes on cell death by controlling salicylic acid signaling
- Author
-
Kohki Yoshimoto
- Subjects
Feedback, Physiological ,Senescence ,Physiological function ,Programmed cell death ,Cell Death ,Acclimatization ,Autophagy ,food and beverages ,Cell Biology ,Plants ,Biology ,Plants, Genetically Modified ,Cell biology ,chemistry.chemical_compound ,chemistry ,Salicylic Acid ,Molecular Biology ,Gene ,Plant Physiological Phenomena ,Salicylic acid ,Plant Diseases ,Signal Transduction - Abstract
It has long been recognized that autophagy in plants is important for nutrient recycling and plays a critical role in the ability of plants to adapt to environmental extremes such as nutrient deprivation. Recent reverse genetic studies, however, hint at other roles for autophagy, showing that autophagy defects in higher plants result in early senescence and excessive immunity-related programmed cell death (PCD), irrespective of nutrient conditions. Until now, the mechanisms by which cells die in the absence of autophagy were unclear. In our study, using biochemical, pharmacological and genetic approaches, we reveal that excessive salicylic acid (SA) signaling is a major factor in autophagy-defective plant-dependent cell death and that the SA signal can induce autophagy. These findings suggest a novel physiological function for plant autophagy that operates via a negative feedback loop to modulate proper SA signaling.
- Published
- 2010
31. Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants
- Author
-
Yasuo Niwa, Masanori Shimizu, Hirokazu Kobayashi, Kohki Yoshimoto, and Takanori Hirano
- Subjects
Light ,Transgene ,Mutant ,Genetic Vectors ,Green Fluorescent Proteins ,Arabidopsis ,Plant Science ,medicine.disease_cause ,Green fluorescent protein ,Protein targeting ,Botany ,Tobacco ,Genetics ,medicine ,Arabidopsis thaliana ,Plastid ,DNA Primers ,biology ,Base Sequence ,fungi ,food and beverages ,Cell Biology ,biology.organism_classification ,Plants, Genetically Modified ,Cell biology ,Luminescent Proteins ,Plants, Toxic ,Organelle biogenesis - Abstract
Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.
- Published
- 1999
32. Chloroplasts are partially mobilized to the vacuole by autophagy
- Author
-
Kohki Yoshimoto and Hiroyuki Ishida
- Subjects
Chloroplasts ,biology ,Arabidopsis Proteins ,Ribulose-Bisphosphate Carboxylase ,Green Fluorescent Proteins ,fungi ,RuBisCO ,Autophagy ,Arabidopsis ,food and beverages ,Cell Biology ,Vacuole ,Photosynthesis ,Cell biology ,Chloroplast ,Cytosol ,Biochemistry ,Vacuoles ,Organelle ,biology.protein ,Chloroplast Proteins ,Molecular Biology - Abstract
Excluding the central vacuole, chloroplasts constitute the largest compartment within the leaf cells of plants and contain approximately 80 percent of the total leaf nitrogen, mainly as proteins. Much of this nitrogen is allocated to the carbon-fixing enzyme in photosynthesis, Rubisco. During senescence, plants can mobilize nitrogen from chloroplasts in older leaves to other organs, such as developing seeds. Whereas bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have recently demonstrated that stroma-targeted green fluorescent protein (GFP), DsRed, and GFP-labeled Rubisco can be mobilized to the vacuole of living cells via Rubisco-containing bodies, in an ATG gene-dependent manner. Our results indicate the presence of a specific autophagic pathway for chloroplast stromal proteins, which does not cause chloroplast lysis. Here, we also discuss the involvement of stroma-filled tubules, stromules, which are important for the structural flexibility of the organelle, on the autophagic transfer of stromal proteins to the vacuole.
- Published
- 2008
33. Several Strategies for Dissecting and Controlling Functions in Plant Cells
- Author
-
Yasuo Niwa, Jen Sheen, Yoshihiro Narusaka, Hirokazu Kobayashi, Mao Sakaiya, and Kohki Yoshimoto
- Subjects
biology ,Transgene ,Gene expression ,food and beverages ,Arabidopsis thaliana ,RNA ,Mutagenesis (molecular biology technique) ,biology.organism_classification ,Molecular biology ,Gene ,Genome ,Green fluorescent protein ,Cell biology - Abstract
The strengths of gene promoters functional in chloroplasts have been evaluated for transient expression by particle bombardment to facilitate engineering of the chloroplast genome of Arabidopsis thaliana. The T7 RNA polymerase-recognized promoter was the most active in transgenic A. thaliana with T7 RNA polymcrase destined for chloroplasts. As a vital reporter of gene expression, an improved gene for green fluorescent protein (GFP) was chemically synthesized and resulted in approx. 100 times higher fluorescence intensity in A. thaliana. To overcome the limitations of experimentally designing of engineered proteins, we employed in vitro random mutagenesis of genes and subsequent selection of their transformants under certain conditions. The strategy was successfully applied to the photosystem II reaction center D1 protein to confer phototolerance on it.
- Published
- 1998
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